Microbial utilization and degradation of alkanes was discovered almost a century ago. Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates. The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenases (BMO) are specialized on gaseous small-chain alkanes (C1 to C4), while medium-chain (C5 to C16) alkane hydroxylation appears to be the domain of the CYP153A6 and AlkB enzyme families.
Conversion of C1 to C4 alkanes to the corresponding alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. (van Beilen, J. B., et. al., 2007, Appl. Microbiol. Biotechnol., 74, 13-21). Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth.
AlkB, particularly AlkB from Pseudomonas putida, is a highly studied medium-chain alkane hydroxylase that typically acts on alkanes containing ten or more carbons, although some accept alkanes as small as five carbons. (Baptist, J. N., et. al., 1963, Biochim. Biophys. Acta, 73, 1-6; Nieder, M., et. al., 1975, J. Bacteriol., 122, 93-98; van Beilen, J. B., 1994, Alkane oxidation by Pseudomonas putida: genes and proteins, Ph.D. thesis, University of Groningen, Groningen, The Netherlands; Kok, M., et. al., 1989, J. Biol. Chem., 264, 5435-5441; Staijen, I. E., 2000, Eur. J. Biochem., 267, 1957-1965; van Beilen, J. B., et. al., 1994, Enzyme Microb. Technol., 16, 904-911; and, van Beilen, J. B., et. al., 2005, J. Bacteriol., 187, 85-91). AlkB selectively oxidizes at the terminal carbon to produce 1-alkanols. However, no protein engineering studies have been conducted on this di-iron integral membrane enzyme; particularly, with respect to whether it can support growth on small-chain alkanes. Nucleotide and amino acid sequences for AlkB from P. putida can be found in, and are hereby incorporated by reference from, the GenBank database under the accession Nos. AJ245436 (SEQ ID NO: 1) and P12691 (SEQ ID NO: 2), respectively.
The AlkB enzymes are one of the main actors in medium-chain length alkane hydroxylation by the cultivated bacteria to date. (van Beilen, J. B., et. al. 2006, Appl. Environ. Microb., 72, 59-65). Recent studies have only been able to generate enzymes that hydroxylate propane and higher alkanes primarily at the more energetically favorable subterminal positions. (Fasan, R., et. al., 2007, Angew. Chem. Int. Ed. Engl., 46, 8414-8418; Xu, F. et. al., 2005, Angew. Chem. Int. Ed., 44, 4029-4032). However, no studies to date have attempted to alter the ability of AlkB enzymes to hydroxylate small-chain alkanes. Highly selective and desirable terminal hydroxylation is difficult to achieve by engineering a subterminal hydroxylase. (Peters, M., et. al., 2003, J. Am. Chem. Soc., 125, 13442-13450).
Previous approaches, some of which are described above, relate to in vitro evolution of hydroxylase enzymes that does not provide the opportunity to screen for improved activity on a specific alkane substrate directly. More specifically, previous approaches do not provide the ability to hydroxylate desirable small-chain alkanes at the terminal position. Further, the prior methods lead to low or no terminal hydroxylation activity and often result in high uncoupling.